Abstract: Frequent pathway mutation involving multiple components of the RNA splicing machinery is a cardinal feature of myeloid neoplasms showing myeloid dysplasia, in which the major mutational targets include U2AF35, ZRSR2, SRSF2 and SF3B1. Among these, SF3B1 mutations were strongly associated with MDS subtypes characterized by increased ring sideroblasts, such as refractory anemia and refractory cytopenia with multiple lineage dysplasia with ring sideroblasts, suggesting the critical role of SF3B1 mutations in these MDS subtypes. However, currently, the molecular mechanism of SF3B1mutation leading to the ring sideroblasts formation and MDS remains unknown. The SF3B1 is a core component of the U2-small nuclear ribonucleoprotein (U2 snRNP), which recognizes the 3′ splice site at intron–exon junctions. It was demonstrated that Sf3b1 null mice were shown to be embryonic lethal, while Sf3b1 +/- mice exhibited various skeletal alterations that could be attributed to deregulation of Hox gene expression due to haploinsufficiency of Sf3b1. However, no detailed analysis of the functional role of Sf3b1 in hematopoietic system in these mice has been performed. So, to clarify the role of SF3B1 in hematopoiesis, we investigated the hematological phenotype of Sf3b1 +/- mice. There was no significant difference in peripheral blood counts, peripheral blood lineage distribution, bone marrow total cellularity or bone marrow lineage composition between Sf3b1 +/+ and Sf3b1 +/- mice. Morphologic abnormalities of bone marrow and increased ring sideroblasts were not observed. However, quantitative analysis of bone marrow cells from Sf3b1 +/- mice revealed a reduction of the number of hematopoietic stem cells (CD34 neg/low, cKit positive, Sca-1 positive, lineage-marker negative: CD34-KSL cells) measured by flow cytometry analysis, compared to Sf3b1 +/+ mice. Whereas examination of hematopoietic progenitor cells revealed a small decrease in KSL cell populations and megakaryocyte - erythroid progenitors (MEP) in Sf3b1 +/- mice, and common myeloid progenitors (CMP), granulocyte - monocyte progenitors (GMP) and common lymphoid progenitors (CLP) remained unchanged between Sf3b1 +/+ and Sf3b1 +/- mice. In accordance with the reduced number of hematopoietic stem cells in Sf3b1 +/- mice, the total number of colony-forming unit generated from equal number of whole bone marrow cells showed lower colony number in Sf3b1 +/- mice in vitro. Competitive whole bone marrow transplantation assay, which irradiated recipient mice were transplanted with donor whole bone marrow cells from Sf3b1 +/+ or Sf3b1 +/- mice with an equal number of competitor bone marrow cells, revealed impaired competitive whole bone marrow reconstitution capacity of Sf3b1 +/- mice in vivo. These data demonstrated Sf3b1 was required for hematopoietic stem cells maintenance. To further examine the function of hematopoietic stem cells in Sf3b1 +/- mice, we performed competitive transplantation of purified hematopoietic stem cells from Sf3b1 +/+ or Sf3b1 +/- mice into lethally irradiated mice together with competitor bone marrow cells. Sf3b1 +/- progenitors showed reduced hematopoietic stem cells reconstitution capacity compared to those from Sf3b1 +/+ mice. In serial transplantation experiments, progenitors from Sf3b1 +/- mice showed reduced repopulation ability in the primary bone marrow transplantation, which was even more pronounced after the second bone marrow transplantation. Taken together, these data demonstrate that Sf3b1 plays an important role in normal hematopoiesis by maintaining hematopoietic stem cell pool size and regulating hematopoietic stem cell function. To determine the molecular mechanism underlying the observed defect in hematopoietic stem cells of Sf3b1 +/- mice, we performed RNA-seq analysis. We will present the results of our biological assay and discuss the relation of Sf3b1 and hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

Abstract: Abstract 782 Recent genetic studies have revealed a number of novel gene mutations in myeloid malignancies, unmasking an unexpected role of deregulated histone modification and DNA methylation in both acute and chronic myeloid neoplasms. However, our knowledge about the spectrum of gene mutations in myeloid neoplasms is still incomplete. In the previous study, we analyzed 29 paired tumor-normal samples with chronic myeloid neoplasms with myelodysplastic features using whole exome sequencing (Yoshida et al., Nature 2011). Although the major discovery was frequent spliceosome mutations tightly associated with myelodysplasia phenotypes, hundreds of unreported gene mutations were also identified, among which we identified recurrent mutations involving STAG2, a core cohesin component, and also two other cohesin components, including STAG1 and PDS5B. Cohesin is a multimeric protein complex conserved across species and is composed of four core subunits, i.e., SMC1, SMC3, RAD21 and STAG proteins, together with several regulatory proteins. Forming a ring-like structure, cohesin is engaged in cohesion of sister chromatids in mitosis, post-replicative DNA repair and regulation of gene expression. To investigate a possible role of cohesin mutations in myeloid leukemogenesis, an additional 534 primary specimens of various myeloid neoplasms was examined for mutations in a total of 9 components of the cohesin and related complexes, using high-throughput sequencing. Copy number alterations in cohesin loci were also interrogated by SNP arrays. In total, 58 mutations and 19 deletions were confirmed by Sanger sequencing in 73 out of 563 primary myeloid neoplasms (13%). Mutations/deletions were found in a variety of myeloid neoplasms, including AML (22/131), CMML (15/86), MDS (26/205) and CML (8/65), with much lower mutation frequencies in MPN (2/76), largely in a mutually exclusive manner. In MDS, mutations were more frequent in RCMD and RAEB (19.5%) but rare in RA, RARS, RCMD-RS and 5q- syndrome (3.4%). Cohesin mutations were significantly associated with poor prognosis in CMML, but not in MDS cases. Cohesin mutations frequently coexisted with other common mutations in myeloid neoplasms, significantly associated with spliceosome mutations. Deep sequencing of these mutant alleles was performed in 19 cases with cohesin mutations. Majority of the cohesin mutations (16/19) existed in the major tumor populations, indicating their early origin during leukemogenesis. Next, we investigated a possible impact of mutations on cohesin functions, where 17 myeloid leukemia cell lines with or without cohesin mutations were examined for expression of each cohesin component and their chromatin-bound fractions. Interestingly, the chromatin-bound fraction of one or more components of cohesin was substantially reduced in cell lines having mutated or defective cohesin components, suggesting substantial loss of cohesin-bound sites on chromatin. Finally, we examined the effect of forced expression of wild-type cohesin on cell proliferation of cohesin-defective cells. Introduction of the wild-type RAD21 and STAG2 suppressed the cell growth of RAD21- (Kasumi-1 and MOLM13) and STAG2-defective (MOLM13) cell lines, respectively, supporting a leukemogenic role of compromised cohesin functions. Less frequent mutations of cohesin components have been described in other cancers, where impaired cohesion and consequent aneuploidy were implicated in oncogenic action. However, 23 cohesin-mutated cases of our cohort had completely normal karyotypes, suggesting that cohesin-mutated cells were not clonally selected because of aneuploidy. Alternatively, a growing body of evidence suggests that cohesin regulate gene expression, arguing for the possibility that cohesin mutations might participate in leukemogenesis through deregulated gene expression. Of additional note, the number of non-silent mutations determined by our whole exome analysis was significantly higher in 6 cohesin-mutated cases compared to non-mutated cases. Since cohesin also participates in post-replicative DNA repair, this may suggest that compromised cohesin function could induce DNA hypermutability and contribute to leukemogenesis. In conclusion, we report a new class of common genetic targets in myeloid malignancies, the cohesin complex. Our findings highlight a possible role of compromised cohesin functions in myeloid leukemogenesis. Disclosures: Haferlach: MLL Munich Leukemia Laboratory: Equity Ownership. Alpermann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

Abstract: Abstract 1706 The recent study of whole-exome sequencing on MDS has revealed frequent and specific pathway mutations involving multiple components of the RNA splicing machinery, including U2AF35, SRSF2, SF3B1 and ZRSR2. The mutually exclusive manner of these mutations among MDS cases also supported that deregulated RNA splicing contributes to the pathogenesis of MDS. Interestingly, the distribution of these splicing pathway mutations shows a substantial difference with regard to disease subtypes. Thus, the SF3B1 mutations are by far the most frequent in RARS and RCMD-RS cases, and the SRSF2 mutations are more prevalent in CMML. SRSF2 is a member of the SR protein family that is commonly characterized by one or two RNA recognition motifs (RRM) and a signature serine/arginine-rich domains (RS domains). The SR proteins interact with other spliceosome components through their RS domains, among which most extensively characterized are SRSF1 (ASF/SF2) and SRSF2 (SC35). Both SR proteins bind a splicing enhancer site within the 3' target exon and also interact with the U2AF, playing an indispensable role in both constitutive and alternative splicing in most cell types. In fact, the knockout of these genes in mice results in embryonic lethality. There is emerging evidence that establishes a connection between the abnormal expression of SR proteins and the development of cancer, mainly as a result of change in the alternative splicing patterns of key transcripts. Increased expression of SR proteins usually correlates with cancer progression, as shown by elevated expression of SR proteins in ovarian cancer and breast cancer. In spite of the similarity in their functions, both proteins are thought to have distinct roles, especially in the pathogenesis of myeloid malignancies, since we found no SRSF1 mutations among 582 cases with myeloid neoplasms. On the other hand, studies have shown that increased expression of SRSF1 transforms immortal rodent fibroblasts and leads to the formation of sarcomas in nude mice, supporting the notion that SRSF1 is a proto-oncogene, whereas SRSF2 does not have transforming activity, indicating a highly specific role of SRSF1 in this type of cancer. Thus, little is known about the biological mechanism by which the SRSF2 mutations are involved in the pathogenesis of MDS, although the mutations at the P95 site are predicted to cause a significant displacement of the RS domain relative to the domain for RNA binding. So to gain an insight into the functional aspect of SRSF2 mutations, we performed sequencing analysis of mRNAs extracted from mutant (P95H) SRSF2-transduced HeLa cells in which expression of the wild-type and mutant SRSF2 were induced by doxycycline. The abnormal splicing in mutant SRSF2-transduced cells was directly demonstrated by evaluating the read counts in different fractions. Next, to investigate functional role of SRSF2 mutant, HeLa cells were transduced with lentivirus constructs expressing either the P95H SRSF2 mutant or wild-type SRSF2, and cell proliferation was examined. After the induction of gene expression, the mutant SRSF2-transduced cells showed reduced cell proliferation. In addition, we transduced P95H SRSF2 constructs into factor-dependent 32D cell lines. The expression of mutant SRSF2 protein resulted in increased apoptosis in the presence of IL-3 and also suppression of cell growth in the presence of G-CSF, which may be related to ineffective hematopoiesis, a common feature of MDS. To further clarify the biological effect of SRSF2 mutants in vivo, a highly purified hematopoietic stem cell population (CD34-c-Kit+ScaI+ Lin-) prepared from C57BL/6 (B6)-Ly5.1 mouse bone marrow was retrovirally transduced with either the mutant or wild-type SRSF2 with EGFP marking. The transduced cells were mixed with whole bone marrow cells from B6-Ly5.1/5.2 F1 mice, transplanted into lethally irradiated B6-Ly5.2 recipients, and we are now monitoring the ability of these transduced cells to reconstitute the hematopoietic system and other hematological phenotypes. Much remains, however, to be unrevealed about the functional link between the abnormal splicing of RNA species and the phenotype of myelodysplasia. Further functional studies should be warranted to understand these mechanisms in detail. In this meeting, we will present the results of our functional studies on the SRSF2 mutations and discuss the pathogenesis of MDS in terms of the alterations of splicing machinery. Disclosures: No relevant conflicts of interest to declare.

Abstract: Abstract 1282 Emerging evidence is establishing a connection between MDS and spliceosome mutations. Spliceosome including SF3b1, U2AF1 and SRSF2 are frequently and exclusively mutated in myelodysplastic syndromes (MDS) and related myeloid neoplasms. Spliceosome mutations occur at varying frequencies in different disease subtypes. SF3B1 was shown to be highly associated with MDS characterized by increased ring sideroblasts and SRSF2 mutations are more prevalent in chronic myelomonocytic leukemia. In spite of the fact that the recent discovery constitutes a novel class of genomic lesions and defines an entirely new pathogenic pathway of leukaemogenesis, the pathogenesis of spliceosome mutation is not largely understood. To understanding the biological consequences of spliceosomal mutations, we previously reported mutant U2AF1 cause altered RNA splicing, and overexpressed mutant U2AF1 decrease in cell proliferarion. However, currently, no functional analysis of SRSF2 mutation has been published. SRSF2 belongs to the serine/arginine-rich (SR) protein family. SR proteins are a family of RNA binding proteins characterized by one or two RNA recognition motifs (RRMs) and a signature RS domain enriched with arginine and serine repeats (RS domain).Growing body of evidence suggests that SR protein may be directly involved in the process of carcinogenesis. Gene knockout experiment indicated SRSF2 is involved with specific pathways in regulating cell proliferation and genomic stability during mammalian organogenesis. In neck and head tumor, SRSF2 is frequently overexpressed. And upregulated SRSF2 increases missplicing and downregulates E-cadherin expression, which is an important tumor suppressor gene. Therefore SRSF2 potential function in tumorigenesis is suggested in epithelial cancers. SRSF2 mutations with MDS exclusively occur at P95 within an intervening sequence between RRM and RS domains, indicating a gain-of-function nature of these mutations. So, to clarify the biological role of SRSF2 mutations in leukemogenesis, we evaluated the oncogenic role of SRSF mutations by expressing a mutant SRSF2 allele in Jurkat cells. The cells transduced with a tumor-derived SRSF2 allele showed reduced cell proliferation and increased apoptosis compared to the mock and wild type SRSF2-transduced cells. Next we performed in vitro colony assay using a highly purified hematopoietic stem cell population (CD34-c-Kit+ScaI+ Lin-(CD34-KSL) cells) collected from C57BL/6 (B6)-Ly5.1 mouse that was retrovirally transduced with mock, mutant or wild-type SRSF2 construct. The mutant SRSF2-transduced cells showed reduced cell proliferation compared with mock- or wild-type SRSF2 transduced cells. Subsequently, we conducted bone marrow transplantaion assay. We collected CD34-KSL cells from B6-Ly5.1 mouse, and retrovirally transduce mock, mutant or wild-type SRSF2 construct, each harbouring the EGFP marker gene. And these cells were sorted by EGFP marker, and transplanted with competitor cells (B6-Ly5.1/5.2 F1 mice origin) into lethally irradiated B6-Ly5.2 mice. The wild-type SRSF2-transduced cells showed a lower reconstitution capacity than the mock-transduced cells. On the other hand, the recipients of the cells transduced with the mutant SRSF2 showed lower EGFP-positive cell chimaerism than those of the mock- or the wild-type SRSF2-transduced. Therefore, the mutant SRSF2 was indicated to have a negative effect on cellular proliferation capacity in vitro and in vivo, and a gain-of-function nature of these mutations is suggested. These results are similar to the effect of U2AF1 mutant, which we reported mutant U2AF1 transduced TF-1 and HeLa cells present with a decrease in cell proliferation and hematopoietic stem cells expressing mutant U2AF1 also displayed lower reconstitution capacity by competitive reconstitution assay in mice. So far, the mechanism responsible for the growth advantage of mutant cells in patient is unclear. We furthermore observe hematopoietic phenotype of the bone marrow transplanted model mouse. SRSF2 mutations can coexist with mutations in TET2, ASXL1 and RUNX1. Therefore we performed additionally bone marrow transplantation assay, utilizing hematopoietic cells derived from TET2 knockdown mice, as a model of multistep carcinogenesis. We will present the results of our biological assay on the SRSF2 mutations and discuss the pathogenesis of MDS. Disclosures: No relevant conflicts of interest to declare.

Abstract: Germ line DDX41 variants have been implicated in late-onset myeloid neoplasms (MNs). Despite an increasing number of publications, many important features of DDX41-mutated MNs remain to be elucidated. Here we performed a comprehensive characterization of DDX41-mutated MNs, enrolling a total of 346 patients with DDX41 pathogenic/likely-pathogenic (P/LP) germ line variants and/or somatic mutations from 9082 MN patients, together with 525 first-degree relatives of DDX41-mutated and wild-type (WT) patients. P/LP DDX41 germ line variants explained ∼80% of known germ line predisposition to MNs in adults. These risk variants were 10-fold more enriched in Japanese MN cases (n = 4461) compared with the general population of Japan (n = 20 238). This enrichment of DDX41 risk alleles was much more prominent in male than female (20.7 vs 5.0). P/LP DDX41 variants conferred a large risk of developing MNs, which was negligible until 40 years of age but rapidly increased to 49% by 90 years of age. Patients with myelodysplastic syndromes (MDS) along with a DDX41-mutation rapidly progressed to acute myeloid leukemia (AML), which was however, confined to those having truncating variants. Comutation patterns at diagnosis and at progression to AML were substantially different between DDX41-mutated and WT cases, in which none of the comutations affected clinical outcomes. Even TP53 mutations made no exceptions and their dismal effect, including multihit allelic status, on survival was almost completely mitigated by the presence of DDX41 mutations. Finally, outcomes were not affected by the conventional risk stratifications including the revised/molecular International Prognostic Scoring System. Our findings establish that MDS with DDX41-mutation defines a unique subtype of MNs that is distinct from other MNs.

Abstract: DDX41 was identified as a causative gene for late-onset familial myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). While DDX41 is thought to be one of the most frequent targets of germline mutations responsible for sporadic cases with AML/MDS and other myeloid neoplasms, the entire spectrum of pathogenic DDX41 variants and their effect size therein are still to be elucidated, and so was the clinical picture of DDX41-mutated myeloid neoplasms. In this study, through an international collaboration, we investigated DDX41 variants in a total of 5,609 sporadic cases with different myeloid neoplasms from different ethnicities, using next generation sequencing. Mutations in the major driver genes commonly mutated in AML/MDS were also examined. Frequencies of germline DDX41 variants were compared between sporadic cases with myeloid neoplasms and healthy individuals (n=13,906). We also characterized genetic/clinical features of DDX41-mutated myeloid neoplasms. We identified a total of 208 (3.6%) patients with DDX41 variants, of whom approximately 50% had both germline and somatic mutations, whereas 37% and 13% had either germline or somatic mutations alone, respectively. Somatic mutations were found in 58% of patients with germline mutation, which was significantly higher than those without (0.21%) (P & lt;0.0001). No somatic mutation was identified in healthy individuals. Among 174 germline variants, truncating and missense mutations were found in 93 and 81 cases, respectively, whereas only 1.9% of somatic mutations were truncating (P & lt;0.0001). Among 21 cases with somatic mutations alone, 4 had multiple somatic mutations and an additional 4 had loss-of-heterozygosity of the DDX41 locus (5q35.3), including 3 with uniparental disomy and 1 with deletion. Thus, 8 out of 21 cases with somatic mutation alone were suspected to have biallelic DDX41 mutations. Germline DDX41 variants showed a conspicuous ethnic diversity; the most frequent germline variants were A500fs in Japan, D140fs in USA, Q41* in Germany, G218D in Italy, M1I in Sweden, S21fs in Thailand. The M1I variant was also seen in other European countries, but not in Japan or Thailand, while no A500fs mutation was found in Europe. Among the Japanese population, significant enrichment in myeloid neoplasms was observed not only for truncating variants, such as A500fs (odds ratio (OR)=12.1) and E7X (OR=11.0) but also for missense variants, including Y259C (OR 14.3) and E256K (OR 7.81), frequently accompanied by a somatic DDX41 mutation (Figure 1). Patients with germline and/or somatic DDX41 variants were significantly older than those without (P=0.00076) and more prevalent in male than female (OR=3.14; P & lt;0.0001). DDX41 variants were significantly more frequent in MDS (4.7%) and AML (2.9%), compared with other myeloid neoplasms (0.58%). Among AML, mutations were more frequent in AML with myelodysplasia-related changes (P & lt;0.00001). Patients with MDS having both germline and somatic mutations were more likely to classified in refractory anemia with excess blasts (RAEB), compared with those with germline or somatic alone (P=0.029). DDX41 variants were significantly associated with lower WBC and granulocyte counts. Most frequently co-occurring mutations included those in ASXL1, SRSF2, TET2, CUX1, and DNMT3A, of which only CUX1 mutations were statistically significant. Overall, no difference was observed in overall survival (OS) between DDX41-mutated and unmutated cases. However, among RAEB cases, DDX41 variants were associated with a significantly longer OS (P=0.0039). In summary, the majority of DDX41-mutated cases had a germline variant, although a minority had somatic mutations alone. Pathogenic DDX41 alleles have a large ethnic diversity, where not only truncating variants but also missense variants are associated with an increased risk of the development of myeloid neoplasms. Disclosures Kanda: Chugai Pharma: Honoraria, Research Funding; Merck Sharp & Dohme: Honoraria; Mundipharma: Honoraria; Ono Pharmaceutical: Honoraria; Nippon Shinyaku: Honoraria, Research Funding; Takeda Pharmaceuticals: Honoraria; Alexion Pharmaceuticals: Honoraria; Shire: Honoraria; Mochida Pharmaceutical: Honoraria; Daiichi Sankyo: Honoraria; Shionogi: Research Funding; Meiji Seika Kaisha: Honoraria; Sanofi: Honoraria, Research Funding; Otsuka: Honoraria, Research Funding; Janssen: Honoraria; Pfizer: Honoraria, Research Funding; Eisai: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Sumitomo Dainippon Pharma: Honoraria; Novartis: Honoraria; Kyowa Kirin: Honoraria, Research Funding; Astellas Pharma: Honoraria, Research Funding. Miyazaki:NIPPON SHINYAKU CO.,LTD.: Honoraria; Sumitomo Dainippon Pharma Co., Ltd.: Honoraria; Kyowa Kirin Co., Ltd.: Honoraria; Novartis Pharma KK: Honoraria; Astellas Pharma Inc.: Honoraria; Otsuka Pharmaceutical: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria; Celgene: Honoraria. Maciejewski:Alexion, BMS: Speakers Bureau; Novartis, Roche: Consultancy, Honoraria. Ogawa:Otsuka Pharmaceutical Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; KAN Research Institute, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Asahi Genomics Co., Ltd.: Current equity holder in private company; Chordia Therapeutics, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Eisai Co., Ltd.: Research Funding.

Abstract: Abstract 458 MDS and related disorders comprise a group of myeloid neoplasms characterized by deregulated blood cell production and a predisposition to AML. Although currently, a number of gene alterations have been implicated in the pathogenesis of MDS, they do not fully explain the pathogenesis of MDS, because many of them are also found in other myeloid malignancies and roughly 20% of MDS cases have no known genetic changes. So, in order to clarify a complete registry of gene mutations in MDS and identify those discriminate MDS from other myeloid neoplasms, we performed whole-exome sequencing of 29 cases showing myelodysplasia. A total of 268 somatic mutations or 9.2 mutations per sample were identified. Among these 41 occurred in recurrent gene targets, which not only included a spectrum of known gene targets in MDS, such as TET2, EZH2, NRAS/KRAS, RUNX1, TP53 and DNMT3A, but also affected previously unknown genes that are commonly mapped to the RNA splicing pathway, including U2AF35, SRSF2 and ZRSR2. Together with additional three (SF3A1, SF3B1 and PRPF40B) found in single cases, 16 (55.2%) of the 29 discovery cases carried a mutation affecting the component of the splicing machinery. To confirm the observation, we examined 9 spliceosome genes for mutations in a large set of myeloid neoplasms (N=582) using a high-throughput mutation screen of pooled DNA followed by confirmation/identification of candidate mutations. In total, 219 mutations were identified in 209 out of the 582 specimens of myeloid neoplasms. Mutations of the splicing machinery were highly specific to diseases showing myelodysplastic features, including 19 of 23 (83%) cases with RARS, 43 of 50 (86%) RCMD-RS, 68 of 155 (44%) other MDS, 48 of 88 (55%) CMML, and 16 of 62 (26%) secondary AML with MDS features with a string preference of SF3B1 mutations to RARS and RCMD-RS and of SRSF2 to CMML, while they were rare in cases with de novo AML (N=151) and MPD (N=53). The mutations among 4 genes, U2AF35 (N = 37), SRSF2 (N = 56), SF3B1 (N = 79) and ZRSR2 (N = 23), explained most of the mutations with a much lower mutational rate for SF3A1 (N = 8), PRPF40B (N = 7), U2AF65 (N = 4) and SF1 (N = 5). Interestingly, mutations in the former three genes showed clear hot spots, indicating a gain-of-function nature of these mutations. On the other hand, two thirds of the ZRSR2 mutations are nonsense or frameshift changes causing premature truncation of the protein. Significantly, these mutations occurred in an almost completely mutually exclusive manner among mutated cases, and commonly affected those components of the splicing complex that are engaged in the 3' splice site recognition during RNA splicing, strongly indicating production of unspliced or aberrantly spliced RNA species are incriminated for the pathogenesis of MDS. In fact, when transduced into HeLa cells, the recurrent S34F U2AF35 mutant induced the increase in the production of unspliced RNA species and elicited the activation of the nonsense mediated decay pathway. Functionally, the U2AF35 mutants seemed to cause deregulated stem cell functions, because CD34(−) KSL cells transduced with various U2AF35 mutants invariably showed reduced chimerism in competitive reconstitution assay. In accordance with this, the S34F U2AF35 mutant lead to suppression of cell growth in a variety of cell types, including HeLa cells, in which expression of the mutant induced a G2/M cell cycle arrest and increased apoptosis. In conclusion, whole-exome sequencing unexpectedly revealed the high frequency of the splicing pathway mutations in MDS and related myeloid neoplasms, providing the first evidence indicating that compromised RNA splicing by gene mutations are responsible for human pathogenesis. Disclosures: Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership.